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EnzyLight™ ADP/ATP Ratio Assay Kit

BCA
Catalog No. ABIN1000320
  • Target
    ADP/ATP Ratio
    Application
    Biochemical Assay (BCA)
    Characteristics
    Safe. Non-radioactive assay.
    Homogeneous and convenient. Mix-incubate-measure type assay. No wash and reagent transfer steps are involved.
    Robust and amenable to HTS: Z' factors of 0.5 and above are routinely observed in 96-well and 384-well plates. Can be readily automated on HTS liquid handling systems for processing thousands of samples per day.
    Components
    Assay Buffer: 10 mL. Substrate: 120 µL. Cosubstrate 120 µL. ATP Enzyme: 120 µL. ADP Enzyme: 120 µL.
  • Application Notes
    Apoptosis and Necrosis determination in cells. Cell proliferation: effects of cytokines, growth factor, nutrients.
    Drug discovery: high-throughput screening for anticancer drugs.
    Protocol
    1. ATP Assay. Bring Assay Buffer, Substrate and Cosubstrate to room temperature. Thaw enzyme on ice or at 4°C. Fresh Reconstitution is recommended. Store unused reagents including the enzyme at - 20°C. ATP Reagent. For each 96-well, mix 95 µL Assay Buffer with 1 µL Substrate, 1 µL Cosubstrate and 1 µL ATP Enzyme. Add 90 µL ATP Reagent to each well and mix by tapping the plate. After 1 min, read luminescence (RLU A) on a luminometer.
    2. ADP Assay. Prepare ADP Reagent: for each 96-well, mix 5 µL dH2O with 1 µL ADP Enzyme. Ten minutes after reading the luminescence for ATP (RLU A), read the luminescence of the samples again (RLU B). This measurement provides the background prior to measuring ADP (i.e. the residual ATP signal). Immediately following reading RLU B, add 5 µL ADP Reagent to each well and mix by tapping the plate or pipetting up and down. After 1 min. read luminescence (RLU C) on a luminometer.
    Sample Preparation

    For suspension cells, transfer 10 µL of the cultured cells (10 3 -10 4 ) into a white opaque 96 well plate. Adherent cells: culture 10 3 -10 4 cells in white opaque microplate. At the time of assay, remove the culture medium immediately before adding 100 µL ATP Reagent.

    Restrictions
    For Research Use only
  • Storage
    -20 °C
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  • Target
    ADP/ATP Ratio
    Background
    Quantitative bioluminescent assay for ADP:ATP ratio.
    Procedure: 20 min.

    Changes in the ADP/ATP ratio have been used to differentiate modes of cell death and viability. Increased levels of ATP and decreased levels of ADP signify proliferating cells. Conversely, decreased levels of ATP and increased levels of ADP represent apoptotic or necrotic cells where the decrease in ATP and increase in ADP is much more pronounced in necrosis versus apoptosis. This ADP/ATP Ratio Assay Kit provides a rapid method to measure ADP and ATP levels for the screening of apoptosis, necrosis and cell proliferation in mammalian cells. The assay involves two steps. In the first step, the working reagent lyses cells to release ATP and ADP. In the presence of luciferase, ATP immediately reacts with the Substrate D-luciferin to produce light. The light intensity is a direct measure of intracellular ATP concentration. In the second step, the ADP is converted to ATP through an enzyme reaction. This newly formed ATP then reacts with the D-luciferin as in the first step. This non-radioactive, homogeneous cell-based assay is performed in microplates. The reagent is compatible with all culture media and with all liquid handling systems for high-throughput screening applications in 96-well and 384-well plates.
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